1. Field of the Invention
A light-scattering instrumentation system is described which allows the quantitation of latex agglutination reactions which employ immunochemistry. More particularly, the instrumentation system finds use in antigen-antibody reactions, for example, in therapeutic drug monitoring or for drugs of abuse determination.
2. Description
Latex agglutination reactions are very popular to monitor biochemical substances in biological fluids, such as urine, serum, etc. A large number of pregnancy tests monitoring human chronic gonadotropin (HCG) in urine is available, which employ this agglutination principle. In a simplified manner, the agglutination inhibition reaction can be thought of as a four step process. (1) Free antigen (Ag) from a subject is added to an antibody (Ab) reagent to form complexes. (2) Latex-bonded antigen (L-Ag) is then added to the complexes and allowed to compete to form other complexes with any unreacted antibody (Ab). (3) As the formation of latex-bonded antigen-antibody complexes continues, aggregation begins to take place. In this phase of the reaction, light transmitted through the sample decreases. (4) As the reaction goes to completion, sometimes taking as long as 90 minutes, the agglutination causes clumping of the sample so that there is a dramatic change in the way the sample looks. Furthermore, after this time period the upper portion of the sample appears clear as aggregates fall to the bottom of the reaction container. During this last step, the light transmitted through the clear upper portion of the reaction container begins to increase. Thus, the reaction can be monitored by visually noting the formation or absence of aggregates (floccules). This is the basis of the prior art non-instrumental or manual agglutination testing.
In the prior art, it also was possible automatically to monitor agglutination reactions using either nephelometry or spectrophotometry. Should analytical methodology such as nephelometry or spectrometry be employed, it is possible to determine the course of the reaction quantitatively in less time and to assign a concentration value to the electronic signal generated from the sample and measured at a given time.
With nephelometry, the light scattered by a sample is obtained by measuring with a detector the scattered power at an angle to the incident light beam. By light scattering, it is meant that small particles interact with incident light and reflect secondary light. The intensity of the scattered light is a function of the relative refractive index, the size parameters and the angle of observation relative to the incident beam. Other sample parameters such as shape, absorption, the concentration and size distribution of the particles and optical anisotropism (i.e., intensity of scattered light as a function of the angle of observation) also influence the intensity distribution of scattered light.
Two competing processes occur during the early course of the latex agglutination reaction using nephelometric technigues. These processes effect the course of what happens to the light directed into the sample which is normally contained in solution. As the particles become larger, the direct transmission of light through the sample, at a small acceptance angle, is reduced. This is due to the increase in the size of the particles due to agglutination. However, as the size of the particles increase, there is an increase in the light which is scattered at small forward angles from the light source.
Once the incident light has interacted with the sample, the scattered light is then detected by the instrument. Different detectors have different spectral response characteristics.
Many different types of light sources are used in prior art nephelometers. The monochromatic, collimated laser beam has high radiant energy density which makes it suitable for scattered light measurements in a forward direction.
The prior art manual and automatic agglutination measuring devices suffer from the disadvantages of requiring significant time to conduct the readings and of being able to read only one sample at a time. Moreover, the insensitivity of the systems to small concentrations in small volumes of samples leads to inaccurate readings reducing their applicability to qualitative rather than quantitative uses.